This proposal is designed to develop systems for cloning stable small and large DNA fragments, which also are optimized for producing a series of overlapping subclones, using either the single stranded technique or the double stranded procedure. Vectors will be constructed from existing vectors, using a powerful new insertion technique. Construction success will lead to development and production of integrated kits. Phase I objectives are construction of a vector with a multiple cloning region, optimized for both single and double stranded subcloning procedures, and modifying the vector to includes a cos site to permit up to 40kb fragments to be cloned stably.